Masayuki Kamo

Novel protein fold discovered in the PabI family of restriction enzymes

Although structures of many DNA-binding proteins have been solved, they fall into a limited number of folds. Here, we describe an approach that led to the finding of a novel DNA-binding fold. Based on the behavior of Type II restriction–modification gene complexes as mobile elements, our earlier work identified a restriction enzyme, R.PabI, and its cognate modification enzyme in Pyrococcus abyssi through comparison of closely related genomes. While the modification methyltransferase was easily recognized, R.PabI was predicted to have a novel 3D structure. We expressed cytotoxic R.PabI in a wheat-germ-based cell-free translation system and determined its crystal structure. R.PabI turned out to adopt a novel protein fold. Homodimeric R.PabI has a curved anti-parallel β-sheet that forms a ‘half pipe’. Mutational and in silico DNA-binding analyses have assigned it as the double-strand DNA-binding site. Unlike most restriction enzymes analyzed, R.PabI is able to cleave DNA in the absence of Mg2+. These results demonstrate the value of genome comparison and the wheat-germ-based system in finding a novel DNA-binding motif in mobile DNases and, in general, a novel protein fold in horizontally transferred genes.

A unique catalytic triad revealed by the crystal structure of APE0912, a short-chain dehydrogenase/reductase family protein from Aeropyrum pernix K1.

Short-chain dehydrogenases/reductases (SDRs), about 250-residue long, are enzymes that catalyze NAD(P)(H)-dependent oxidation/reduction of various substrates such as alcohols, sugars, steroids, aromatic compounds, and xenobiotics.1–3 SDR family proteins have the N-terminal Rossman fold made up of 7 to 8 b-strands, which binds the cofactor NAD(P)(H), and a catalytic triad composed of Ser-Tyr-Lys.4 The APE0912 gene of hyperthermophilic archaea Aeropyrum pernix K1 encodes an SDR family protein with an unknown function.5 We have determined the crystal structure of APE0912 at 1.8-Å resolution. The overall structure of APE0912 is similar to those of other SDR family members, but APE0912 does not have a catalytic triad Ser-Tyr-Lys characteristic of the SDR family members. Instead, APE0912 has a unique Ser-Ser-Arg triad. The two molecules in the asymmetric unit exhibit open and closed forms that alter the accessibility of substrates and cofactors to the Ser-SerArg triad. Here, we report the crystal structure of APE0912 with its unique catalytic triad and the structural difference between open and closed forms.

Crystal structure of thioredoxin domain of ST2123 from thermophilic archaea Sulfolobus tokodaii strain7

Thioredoxin (TRX) is a well-known small enzyme that is present in all living organisms. It was first described in 1964 by Laurent et al.1 as a redox protein with a conserved active-site sequence, Trp–Cys– Gly(Ala)–Pro–Cys, that catalyzes many redox reactions through the reversible oxidation of dithiol to a disulfide. TRX has long been employed as a model system for studying protein stability because of its globular shape, small size, and high thermostability.2–5 The open reading frame of ST2123 from thermophilic archaea Sulfolobus tokodaii strain7, consisting of 140 amino acid residues with a molecular mass of 15.7 kDa, was annotated as a hypothetical TRX protein. A PSI-BLAST search, using the amino acid sequence of ST2123 as the query, identified from 37 to 140 amino acid residues, has up to 40% of similarity to the thioreoxins of known structure. Here we report the first crystal structure of archaeal TRX of ST2123 from S. tokodaii at 1.49 Å resolution and discuss the characteristic features of this thermostable protein.

Crystallization and preliminary X-ray analysis of carboxypeptidase 1 from Thermus thermophilus.

Carboxypeptidase 1 from the thermophilic eubacterium Thermus thermophilus (TthCP1, 58 kDa), a member of the M32 family of metallocarboxypeptidases, was crystallized by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals diffracted X-rays to beyond 2.6 A resolution using a synchrotron-radiation source. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 171.0, b = 231.6, c = 124.9 A. The crystal contains three molecules in an asymmetric unit (VM = 2.11 A3 Da(-1)) and has a solvent content of 61.5%.

Preliminary X-ray crystallographic analysis of thermolysin in the presence of 4 M NaCl.

The activity of thermolysin (EC 3.4.24.27) is greatly enhanced by high concentrations of neutral salts. For instance, 4 M NaCl enhances the activity 13-15-fold [Holmquist & Vallee (1976), Biochemistry, 15, 101-107; Inouye (1992), J. Biochem. (Tokyo), 112, 335-340]. To clarify the structural basis of the activation of thermolysin by high concentrations of NaCl, we have developed a new method to introduce 4 M NaCl into the P6(1)22 crystal of thermolysin originally grown without NaCl. The crystal obtained by this method diffracted X-rays to 2.43 A. No unit-cell parameter change was observed except the length of the c axis, which was elongated by 9.6% by the introduction of 4 M NaCl.

Crystallization and preliminary X-ray crystallographic analysis of peptide deformylase from Thermus thermophilus HB8

Peptide deformylase (PDF) is responsible for cleaving the formyl group at the N-terminus of nascent polypeptide chains in eubacteria and is essential to bacterial cell viability. A recombinant PDF of the thermophilic bacterium Thermus thermophilus HB8 has been crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystals belonged to the tetragonal space group P4(1) or P4(3), with unit-cell parameters a = b = 62.58, c = 105.27 A, and are most likely to contain two molecules in an asymmetric unit, giving a crystal volume per protein weight (V(M)) of 2.3 A(3) Da(-1) and a solvent content of 46.7%.